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Cell Cycle-Regulated Oscillator Coordinates Core Histone Gene Transcription Through Histone Acetylation

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Cell Cycle-Regulated Oscillator Coordinates Core Histone Gene Transcription Through Histone Acetylation Cell cycle-regulated oscillator coordinates core histone gene transcription through histone acetylation Christoph F. Kurata,1, Jean-Philippe Lambertb, Julia Petschnigga, Helena Friesena, Tony Pawsonb,c, Adam Rosebrocka, Anne-Claude Gingrasb,c, Jeffrey Fillinghamd, and Brenda Andrewsa,c,2 aThe Donnelly Center, University of Toronto, Toronto, ON, Canada M5S 3E1; bLunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5; cDepartment of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 3E1; and dDepartment of Chemistry and Biology, Ryerson University, Toronto, ON, Canada M5B 2K3 Edited by Jasper Rine, University of California, Berkeley, CA, and approved August 21, 2014 (received for review July 25, 2014) DNA replication occurs during the synthetic (S) phase of the eukary- Although the roster of histone chaperones and chromatin otic cell cycle and features a dramatic induction of histone gene remodelers involved in histone gene regulation is impressive, the expression for concomitant chromatin assembly. Ectopic production cell cycle oscillator responsible for restricting histone gene acti- of core histones outside of S phase is toxic, underscoring the critical vation to S phase has remained elusive. In this context, we de- importance of regulatory pathways that ensure proper expression cided to revisit the functions of two poorly understood proteins of histone genes. Several regulators of histone gene expression in that physically interact and are involved in histone gene regula- the budding yeast Saccharomyces cerevisiae are known, yet the key tion, suppressor of Ty (Spt)10 and Spt21 (15–21). Spt10 is a DNA- oscillator responsible for restricting gene expression to S phase has binding protein that localizes to the upstream-activation sequences remained elusive. Here, we show that suppressor of Ty (Spt)10, a pu- (UAS) of histone genes (22, 23). Spt10 contains a putative HAT tative histone acetyltransferase, and its binding partner Spt21 are key domain similar to that of general control nonderepressible (Gcn) 5 – determinants of S-phase specific histone gene expression. We show that (19, 24), which was reported to be involved in acetylation of H3K56 Spt21 abundance is restricted to S phase in part by anaphase pro- at histone promoters in vivo (14). However, to date, HAT activity of moting complex Cdc20-homologue 1 (APCCdh1) and that it is re- Spt10 has not been demonstrated in vitro. cruited to histone gene promoters in S phase by Spt10. There, Spt21 is a protein of unknown function that physically inter- Spt21-Spt10 enables the recruitment of a cascade of regulators, acts with Spt10 (19). Like Spt10, Spt21 influences histone gene including histone chaperones and the histone-acetyltransferase transcription as spt21Δ mutants display dramatically reduced general control nonderepressible (Gcn) 5, which we hypothesize lead to histone acetylation and consequent transcription activation. levels of HTA2, HTB2, and HHF2 transcripts in logarithmically growing cells (15, 25). Here we show that Spt21 is a cell cycle os- cillator that serves as a master regulator of S-phase–dependent ukaryotic chromosomes are composed of chromatin, which in histone gene expression. We demonstrate that Spt10 is required turn is composed of a fundamental repeated unit of a histone E to establish repression by the recruitment of HIR and HIR- octamer and DNA, the nucleosome. Each histone octamer includes dependent regulators outside of S phase. Furthermore, the ex- two H3-H4 histone dimers flanked on either side by H2A-H2B pression of Spt21 is cell cycle-regulated with levels peaking in dimers. The four replication-dependent (RD) core histones (H2A, S phase, when it is recruited to histone gene promoters by its H2B, H3, and H4) are among the most conserved eukaryotic partner protein Spt10. We use genetic and biochemical experi- proteins, and all characterized eukaryotic genomes carry more than ments to show that the abundance of Spt21 during G1 phase is one gene encoding each core histone protein. Budding yeast con- tains two copies of each RD histone gene, each arranged in Significance opposite orientation to a gene encoding its partner within the nucleosome: HHT1-HHF1 and HHT2-HHF2,thetwogenepairs DNA replication and histone gene transcription are tightly encoding core histones H3 and H4, and HTA1-HTB1 and HTA2- linked and occur during the S phase of the eukaryotic cell cycle. HTB2, the two gene pairs encoding H2A and H2B (for recent Histone production outside of S phase is highly toxic, under- reviews see refs. 1 and 2). scoring the importance of regulatory pathways that control Histone gene expression is repressed outside of the synthetic histone gene expression. Although various histone regulators (S) phase by a histone chaperone called the histone regulatory (HIR) have been discovered, the molecular mechanisms responsible complex, which is recruited to a poorly defined DNA sequence, the for the spatial and temporal control of histone gene expression negative regulatory (NEG) region, present upstream of three of the have remained elusive. Here, we describe the discovery of four histone-gene pairs in yeast: HTA1-HTB1, HHT1-HHF1,and Spt21 as a long-elusive cell cycle oscillator responsible for HHT2-HHF2 (3–7). HTA2-HTB2, the fourth histone gene pair, does restricting histone gene transcription to the S phase of the not have a NEG region and is regulated in a HIR-independent eukaryotic cell cycle. We show here that Spt21, together with manner. At HIR-dependent promoters, HIR recruits other histone its partner protein Spt10, regulates histone gene transcrip- chaperones, Asf1 and Rtt106, as well as the chromatin boundary tion by enabling the recruitment of a roster of chromatin- protein Yta7 (8, 9) and the remodel structure of chromatin remodeling proteins. (RSC) ATP-dependent chromatin-remodeling complex (10), which together assemble repressive chromatin, blocking recruitment of Author contributions: C.F.K., J.F., and B.A. designed research; C.F.K., J.-P.L., J.P., and H.F. performed research; T.P., A.R., and A.-C.G. contributed new reagents/analytic tools; C.F.K., RNAPII (11, 12). In S phase, this repressive chromatin is overcome, J.-P.L., J.P., H.F., and A.R. analyzed data; and C.F.K., J.F., and B.A. wrote the paper. allowing recruitment of RNAPII and activation of transcription. The authors declare no conflict of interest. After recruitment of RNAPII, the AAA-ATPase Yta7 is important This article is a PNAS Direct Submission. for efficient transcript elongation by evicting histones H3-H4 (11–13). 1Present address: Cancer Research UK London Research Institute, Clare Hall Laboratories, Other chromatin remodelers also have roles in histone gene South Mimms EN6 3LD, United Kingdom. activation, including the SWI/SNF chromatin-remodeling com- 2To whom correspondence should be addressed. Email: [email protected]. plex, which activates NEG-dependent histone genes (14), and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the histone-acetyltransferase (HAT) complex Rtt109-Vps75 (8). 1073/pnas.1414024111/-/DCSupplemental. 14124–14129 | PNAS | September 30, 2014 | vol. 111 | no. 39 www.pnas.org/cgi/doi/10.1073/pnas.1414024111 Downloaded by guest on September 25, 2021 regulated by the anaphase-promoting complex/cyclosome (APC/C) A associated with its activator protein Cdc20-homologue 1 (Cdh1). During S phase, Spt21 accumulates and recruits the Gcn5 HAT to histone gene promoters, where they influence histone gene transcription. Our data reveal an important cell cycle oscillator that links the cell cycle machinery and histone acetylation and explain how the timing of histone acetylation at histone gene B promoters leads to gene activation. C Results Spt10 and Spt21 Recruit HIR and Associated Proteins/Complexes. To explore the mechanism of Spt10-dependent activation of histone gene transcription, we first used affinity purification and mass spectrometry to discover proteins associated with Spt10. Spe- cifically, we used a tandem affinity purification (TAP)-tagged version of Spt10 expressed either at its endogenous locus or from an inducible promoter (GAL1-10) and a modified chromatin immunoprecipitation (mChIP) protocol to identify proteins associated with Spt10 on chromatin (8, 11, 26). We discovered interactions between Spt10 and all HIR subunits (Hir1, Hir2, Hir3, and Hpc2), RSC, as well as most subunits of SWI/SNF (Fig. S1). We did not identify peptides for these Spt10-interacting proteins across several control purifications, and the recovery of peptides for interacting proteins scaled with the amount of Spt10 in our purifications. We performed a similar mChIP analysis with Spt21-TAP and detected SWI/SNF peptides, but only when Spt21 was overexpressed, consistent with its low abundance relative to Spt10 (Fig. S1). We also confirmed the Spt10/Spt21 interaction described previously (Fig. S1)(19). Because we detected all four HIR subunits in our mChIP of Spt10-TAP, we next used chromatin immunoprecipitation (ChIP) to test whether SPT10 is required for recruitment of HIR (Hir1- TAP), the HIR-dependent regulators RSC (Rsc8-TAP), SWI/SNF (Snf6), Rtt106-TAP, and Asf1-TAP, and the transcriptional acti- vator Yta7 (Yta7-TAP) (8, 11, 13) to the promoter region of Fig. 1. The Spt10/Spt21 complex recruits chromatin-modifying proteins and HTA1-HTB1 (Fig. 1A) (11). Consistent with an important function complexes to the HTA1-HTB1 promoter. (A) Schematic representation of the for the Spt10–HIR interaction on chromatin, we observed a strong HIR-dependent HTA1-HTB1 and HIR-independent HTA2-HTB2 histone gene reduction in the recruitment of both negative regulators of histone promoters. UAS sequences (green circles) found in both regulatory regions gene expression—HIR, Rtt106, Asf1, and RSC–as well as activa- (for a comprehensive review see ref. 2). Primer pairs used for ChIP assays are tors of histone transcription–SWI/SNF and Yta7–to the HTA1- shown with black arrows (8, 10, 11). (B) Spt10 and Spt21 recruit histone HTB1 promoter in an SPT10 deletion strain (Fig.
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